The purpose of this proposal is to identify and study the function of components of the mitotic spindle at the molecular level. Although tubulin is the most abundant component of the spindle, it serves primarily a structural role. The activity of microtubules is likely to be understood through the molecules that govern their assembly and interaction with poles, chromosomes, the midbody and perhaps other spindle structures. This proposal will focus on specific molecules recently identified as components of the spindle. As determined by reaction with monoclonal antibodies specific for a phosphorylated epitope (MPM), a subset of these components has been shown to become phosphorylated at mitosis and dephosphorylated upon the return to interphase. The phosphorylation/dephosphorylation of specific spindle components may serve important regulatory functions for spindle formation, chromosome movement and spindle disassembly. This proposal seeks to determine the MPM-reactive phosphoprotein composition of mitotic MTOCs as well as to identify important non-MPM reactive components; to define biochemically the phosphorylated epitope, to determine the cellular distribution of identified spindle molecules in the dephospho as well as phospho forms in interphase as well as mitosis; to assay the function of phosphorylation/dephosphorylation events in vitro by assaying the effect of phosphorylation on the microtubule nucleating capacity of centrosomes and in vivo by microinjection of phosphorylation/dephosphorylation inhibitors; to test the function of selected spindle components by microinjection of antibodies; to begin the purification of putative protein kinase(s) and phosphorprotein phosphatase(s) responsible for the events; and finally, to attempt to identify and clone genes for selected spindle molecules. These basic studies on cell division may reveal control mechanisms important for understanding the biochemical defects which result in the uncontrolled division of cancer cells.